The present disclosure is directed to means useful in processes of setting up and processing biochemical or molecular biological reactions, and monitoring the status thereof. The preparatory work with respect to a biochemical or molecular biological reaction particularly comprises steps of assembling all necessary reactants of a desired reaction. Typically, a biochemical or molecular biological reaction occurs in aqueous solution. Thus, the different reactants are firstly provided as separate components. They have to be combined to form a reaction mixture, i.e. to form a composition with selected ingredients in prescribed concentrations and under defined other conditions, in order to later on allow the desired reaction to take place. The components are mixed to form the desired composition in aqueous solution. To this end, components can be added to the mixture as dry substances or as already dissolved matter. Also, several components can separately be put together, dissolved and mixed to form a reagent, and several reagents can then be combined and mixed in order to form the desired composition (reaction mixture) for the desired reaction.
Reaction mixtures are typically combined from different aqueous solutions with no obvious (e.g. visible) distinctive features. The skilled person is therefore confronted with the task of carefully monitoring the process of assembling in a reaction vessel or plurality of reaction vessels all the necessary reactants (components, component solutions) of the desired reaction mixture. For example, a skilled lab operator relies on information provided by the markings of the container in which a particular reagent stock is provided. But once a reagent is removed from the container and transferred into the reaction vessel, the physical connection of the reagent and the respective marking is lost. Without any further tracking measures the knowledge about presence and identity of the respective reagent in the reaction vessel is lost. In this context a tracking measure can be a specific record indicating that transfer of an aliquot of a particular reagent stock has been made into a particular reaction vessel. Another example for a tracking measure is a colorant that is present in a particular reagent and that can be identified by optical means such as visual inspection. Once the reagent with the colorant has been added to a reaction vessel, the colorant indicates the presence of the respective reagent in the vessel. This principle is already being used in biochemistry and molecular biology, particularly in applications making use of the polymerase chain reaction (PCR).
As reported in “7.3 Taq DNA polymerase and its modifications” in: Pelt-Verkuil, Elizabeth van, Belkum, Alex van, Hays, John P. (2008) “Principles and Technical Aspects of PCR Amplification”, Springer Netherlands (ISBN 978-90-481-7579-6), some manufacturers supply thermostable DNA polymerase or ready-made PCR master mixes that contain a reportedly inert red dye which does not interfere with either polymerase activity, purification of the amplified PCR product, sequencing or further downstream processing applications. Commercially available examples of these dye related products include REDAccuTaq®, REDTaq® (Sigma) and Red Hot® Taq DNA Polymerase (Thermo Fisher), and others. Such products serve as a tracking measure in that they allow the actual addition and mixing of polymerase or master mixes to be visually controlled. In addition, they facilitate visual tracking of the PCR amplification product on gel electrophoresis. In effect, these dyes act as a molecular weight marker corresponding to a 125 base pair DNA fragment. Another dye advertised as in inert colorant for the purpose of visibly indicating the presence or absence of a reagent in a vial is VisiBlue™ (TATAA Biocenter). WO2015/054396 discloses visible dye formulations as colorants for use with a qPCR master mix on real-time PCR instruments.
Reaction mixtures for PCR are examples for a biochemical or molecular biological reaction that after set-up is started by applying a temperature shift. More generally, concerning the processes of setting up biochemical or molecular biological reactions, the practitioner desires conditions under which the components of a reaction mixture maintain a tendency (i) to be chemically stable, (ii) to not react with each other and (iii) to not become dysfunctional with respect to the desired reaction to be performed later on. Well-known and frequently applied conditions to this end include low ambient temperatures such as temperatures in the range of 0° C. and 4° C. which provide such desired conditions for many biochemical reactants. In particular such low temperatures can be advantageous to inhibit biological activity of enzymes that originate from mesophilic organisms. However, many enzymes from thermostable organisms can be handled at room temperature as their catalytic activity unfolds at higher temperatures. Thus, a large number of preparatory processes of setting up biochemical or molecular biological reactions take place at a lower temperature than the temperature(s) at which the respective reaction proper is performed.
Technically, it is desired to have coloring agent as a marker component of a first liquid composition that would otherwise be indistinguishable from other liquid compositions the first composition could be confused with. Such a coloring agent would advantageously serve as a visual control in a manually performed process of combining different reagents in a reaction mixture. Ideally, such a visual control allows verification whether or not a particular reagent is already present in the mixture. Apart from visual control, a coloring agent is desired which is amenable to assessment by other optical means, particularly by means that can be automated.
In view of the fact that the purpose of the mixture is to allow a biochemical reaction to occur, the coloring agent is further desired to be inert with respect to the biochemical reaction; that is to say, the colorant should not inhibit or otherwise disadvantageously interfere with the biochemical reaction to be performed with the mixture.
In the particular case that during the course of the biochemical reaction any optical properties of the reaction mixture are subject to change, detection of such change is desired not to be disadvantageously affected by the colorant. This is a very specific requirement in case the biochemical reaction to be performed is real-time PCR that includes the step of monitoring fluorescent light emission during the thermocycling steps. A particular challenge in this regard is to find a coloring agent that does not quench fluorescence of one or more fluorescent dye(s) that might be present in the reaction mixture as part of a detection system indicating progress and/or result of the PCR process. Yet, another technical objective is given by the desire to combine visual control with automated control using a photometric readout that can be generated by a device such as, but not limited to, a thermocycler for real-time PCR.
With a more general focus on biochemical reactions that include a temperature shift the authors of the present disclosure took the approach of searching for thermolabile dyes that either have a color that is easily visible to the human eye, even at low concentrations, or dyes that are capable of exhibiting fluorescence. The search specifically focused on dyes that are thermolabile to minimize any influence on photometric detection that may be the case in the course of the processing of the reaction mixture the dye may be comprised in.